Field of the Invention
The present invention relates to a mutant microbial host cell which has been modified, preferably in its genome, to result in a deficiency in the production of a polypeptide having amylase activity, to a method to produce the mutant microbial host cell and to a method to produce a compound of interest using said mutant microbial host cell.
Description of Related Art
An ever increasing number of products is produced by microbial fermentation at industrial scale. Such products range from primary and secondary metabolites, such as e.g. citric acid and antibiotics, respectively, to proteins, enzymes and even complete microorganisms, e.g. in the form of bakers yeast or biomass.
Different host cell types may be used for different production processes. For example: mammalian cell lines are used for antibody production; fungal cells are preferred organisms for production of polypeptides and secondary metabolites; bacterial cells are preferred for small metabolite and antibiotic production; and plant cells are preferred for taste and flavor compounds. An important aspect in the production of valuable products by microbial fermentation is optimization of the productivity of the microbial cells.
Recombinant techniques are widely employed for optimization of the productivity of such cells and/or the processes in which they are used. This can involve a multitude of options.
Some techniques will aim at the over expression of a gene of interest coding for the product or coding for a compound related to the production of the product by the host cell. Gene expression can be modulated in several ways. For example the gene of interest can be placed in the host cell under the expression control of a strong promoter, or it can be placed under the control of a promoter activated by a transcriptional activator which activator can be up-regulated or down-regulated. In yet an alternative approach gene expression can be improved by increasing the copy number of the gene of interest in the host cell used to express the gene. Yet other approaches aiming at improving the productivity of a compound of interest by a host cell can involve deletion or inactivation of competing pathways, changing compartmentalization of enzymes, increasing protein or metabolite secretion, increasing organelle content and the like.
Another important aspect in the production of valuable products by microbial fermentation is the quality and purity of said products after the fermentation and in the final product formulation. These will depend on the specific product quality demands and the final application of the product. Most commercial products produced by microbial fermentation have requirements in their physical appearance (color, odor). Also impurities related to the process (e.g., the presence of unfermented sugar from the media) and the microorganism used (e.g., metabolites produced during the fermentation) can influence the quality of the product. Dealing with these issues requires incorporating purification steps after recovery of the product from the fermentation broth.
If the valuable product is secreted into the nutrient medium, the product can be isolated directly from the medium. If the product is not secreted, it can be isolated from cell lysates. The product may be isolated from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. A variety of procedures known in the art including, but not limited to, chromatography (e. g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e. g., ammonium sulfate precipitation), or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) can be used at this purpose. The use of several purification steps however will increase production costs. In case the product is an enzyme the presence of other enzyme activities, derived from the production microorganism, should be avoided as it can interfere with the application or, more commonly, negatively affect the product stability.
There is therefore a need to develop microorganisms used to express useful products that combine the capacity for expression of commercial quantities thereof while being deficient in the production of enzymes that can complicate recovery, downstream processing or compromise final purity of the products and/or be detrimental in product applications.